Pulmonary arterial hypertension (PAH) is a frequently fatal disorder disproportionately affecting young and middle-aged women. The illness is characterized by progressive shortness of breath, worsening hypoxemia, and ultimately right heart failure. Current vasodilatory therapies provide only modest symptomatic improvement, and new therapeutic modalities are urgently needed. The proposed project investigates a novel mechanobiological feedback mechanism in PAH pathogenesis. A better understanding of the maladaptive and progressive vascular response to pulmonary arterial (PA) stiffening could provide new insights and yield powerful new therapeutic targets for this disease. Pathologically, persons suffering from PAH develop hyperproliferation of apoptosis-resistant pulmonary artery endothelial cells (PAECs) and smooth muscle cells (PASMCs), which leads to progressive PA remodeling. The resulting PA stiffness is independently associated with increased mortality in PAH patients; however, its role in the pathogenesis of PAH has not been fully elucidated. Using atomic force microscopy (AFM), we have determined via work-in-progress that PA matrix stiffening occurs at the micro-scale level in human PAH tissue. In rodent models of PAH, progressive matrix stiffening begins at early time points, weeks before PA pressure begins to rise. In vitro, human PASMCs grown on substrates with the stiffness of remodeled vessels develop increased proliferation and pathogenic alterations in the expression of vasoactive mediators. Taken together, our findings suggest that early development of PA stiffness may fundamentally bias cellular behavior towards progressive vascular remodeling. We hypothesize that increased matrix stiffness in early PAH triggers a local mechanobiologic feedback loop that amplifies vascular remodeling and accelerates disease progression. This study focuses on elucidating the cellular consequences of increased PA stiffness and the mechanisms underlying stiffness-induced changes. In Specific Aim 1, we will determine the phenotypic sequelae of PASMC and PAEC growth on pathologic matrix stiffness, and assess the potential reversibility of the process. In Specific Aim 2, we will investigate the role of an essential mechanotransduction pathway in driving the progressive remodeling response to matrix stiffening. Completion of the proposed aims is expected to lead to further discoveries exploring the role of the mechanical microenvironment in PAH. The NRSA funds will provide two years of research support for Dr. Paul Dieffenbach. He will undertake this project within the Division of Pulmonary and Critical Care Medicine at Brigham and Women's Hospital. He will be working under the close mentorship of his sponsor, Dr. Laura Fredenburgh, and co-sponsor, Dr. Mark Perrella, and will also benefit from the expertise of his collaborators and scientific advisory committee. This project comprises the core of his research fellowship and provides essential training for his career development as an investigator in the field of pulmonary mechanobiology.